Purification of enzymatically inactive peptidylarginine deiminase type 6 from mouse ovary that reveals hexameric structure different from other dimeric isoforms

The murine peptidylarginine deiminase (PAD) has five isoforms encoded by different genes and participates in a variety of cellular functions through the citrullination of target proteins. The crystal structure of human PAD4 with a dimeric form was previously solved because of the enzyme’s relevance to rheumatoid arthritis. PAD6, abundant in mouse oocytes and eggs, is believed to take part in early events of embryogenesis, but its biochemical properties are little understood. Here we have purified and characterized a recombinant PAD6. A PAD6 cDNA was cloned from mouse ovary RNA and expressed in Escherichia coli through pET29 and pGEX vectors. When benzoyl-L-arginine ethyl ester was used as a substrate, no appreciable activity was detected with a cell homogenate under conditions where a human PAD4 cDNA caused significant activity. Both proteins were affinity-purified to near homogeneity. The circular dichroism spectra of PAD6 and human PAD4 were similar in the far ultraviolet region. On molecular sieving, PAD6 was eluted faster than human PAD4. The cross-linking of PAD6 with dimethyl suberimidate clearly showed six bands on an sodium dodecyl sulfate-polyacrylamide gel. These results indicate that PAD6 can constitute a hexameric structure. The purified PAD6 still showed no enzymatic activity. This unique structure and loss in enzymatic activity is strongly suggested to favor the formation of egg cytoplasmic sheets as the architectural protein.

Cryo-EM Data Statistics and Theoretical Analysis of KaiC Hexamer

Cryo-electron microscopy(cryo-EM) provides a powerful tool to resolve the structure of biological macromolecules in natural state. One advantage of cryo-EM technology is that different conformation states of a protein complex structure can be simultaneously built, and the distribution of different states can be measured. This provides a tool to push cryo-EM technology beyond just to resolve protein structures, but to obtain the thermodynamic properties of protein machines. Here, we used a deep manifold learning framework to get the conformational landscape of Kai C proteins, and further obtained the thermodynamic properties of this central oscillator component in the circadian clock by means of statistical physics.

Excitation energy transfer mechanism in C-phycocyanin hexamer

Excitation energy transfer processes and mechanism in C-PC hexamer have been studied in detail by picosecond time-resolved fluorescencs isotropic and anisotropic spectroscopy methods. The experimental results show that there are two types of principal channels, with large probability or amplitude, for linking two trimers viam?m and s?s energy transfer pathways, the energy-transfer time constants of them are about 20 and 10 ps, respectively. Indeed, there exists the evidence for energy-transfer channels of s?f steps in the same monomer and threef?f steps in the same trimer of the C-PC hexamer unit, with small probability or amplitude, and the time constants of them might be ca. 50 and 45 ps separately. Also, the present results show that the hexamer possesses an optimal structure for energy-transfer and for the first time confirm that the dominant energy-transfer processes except those between 1 m?2f, 2m?3f and 3m?1f and so on, in isolated C-PC hexamer, could be described by F?rster dipole-dipole resonance mechanism.

Structure and Magnetic Properties of a Bipyridine-bridged One-dimensional Ni(Ⅱ) Coordination Polymer

A polymer, [NiL（bipy）·2H2O]n（1, bipy = 4,4ˊ-bipyridine）, has been acquired by the reaction of Ni（OAc）2·4H2O with N,Nˊ-bis（2-hydroxyl-5-fluorobenzyl）-2-hydrxylpropane dii- mine in the presence of 4,4ˊ-bipyridine. X-ray crystallographic analysis shows that it is comprised of a mononuclear Ni（Ⅱ） complex [NiL] linked by 4,4ˊ-bipyridine. Its crystal crystallizes in the orthorhombic system, space group C2/c with a = 22.595（3）, b = 18.395（2）, c = 15.434（2） , V = 6155（1）3, Dc = 1.259 g/cm-3, Z = 8, Mr = 583.23, F（000） = 2416.0, μ（MoKa） = 0.681 mm-1, S = 1.049, R = 0.0512 and wR = 0.1108 for 6032 observed reflections（I 〉 2σ（I））. There are water hexamers in the complex. A weak overall ferromagnetic behavior has been evaluated by Curie-Weiss rule and by the formula of alternating ferro-ferromagnetic coupling in one-dimensional Ni（Ⅱ） chain, which gives g = 2.005（1） and J1 = 0.31（3） cm-1.

Liquid Water Structure from Anomalous Density under Ambient Condition

From discussion of the structure of liquid water, we deduce that water under ambient condition is mainly composed of ice Ih-like molecular dusters and clathrate-like molecular clusters. The water molecular dusters remain in a state of chemical equilibrium （reversible clustering reactions）. This structural model can be demonstrated by quantitative study on anomalous density with increasing temperature at ambient pressure.

通过在线7Li-NMR谱对负离子聚合见解的进一步验证

通过对正丁基锂(n-BuLi)/四氢呋喃(THF)引发α-甲基苯乙烯(mSt)负离子本体聚合,验证了n-BuLi缔合体可以引发聚合,形成超分子团聚体,然后在进一步聚合过程中超分子解离.证实了先前提出的负离子聚合的引发机理.通过7Li-NMR对聚合过程的在线检测,进一步证实了mSt在氘代苯为溶剂,THF为调节剂下的负离子聚合以及异戊二烯在非极性条件下的溶液聚合都存在引发剂多元缔合体向二元缔合体转变.研究还发现,少量THF可能使n-BuLi的六元缔合结构2～3个进一步串联起来,但先于六元缔合结构解离.此外,THF与n-BuLi作用,随着n-BuLi/THF的摩尔比从1∶1到1∶5的变化,可以使n-BuLi的巨大缔合体解离并向六元缔合体转变.

Human Endogenous Retrovirus Type W Envelope from Multiple Sclerosis Demyelinating Lesions Shows Unique Solubility and Antigenic Characteristics

In multiple sclerosis(MS),human endogenous retrovirus W family(HERV-W)envelope protein,pHERV-W ENV,limits remyelination and induces microglia-mediated neurodegeneration.To better understand its role,we examined the soluble pHERV-W antigen from MS brain lesions detected by specific antibodies.Physico-chemical and antigenic characteristics confirmed differences between pHERV-W ENV and syncytin-1.pHERV-W ENV monomers and trimers remained associated with membranes,while hexamers self-assembled from monomers into a soluble macrostructure involving sulfatides in MS brain.Extracellular hexamers are stabilized by internal hydrophobic bonds and external hydrophilic moieties.HERV-W studies in MS also suggest that this diffusible antigen may correspond to a previously described highmolecular-weight neurotoxic factor secreted by MS B-cells and thus represents a major agonist in MS pathogenesis.Adapted methods are now needed to identify encoding HERV provirus(es)in affected cells DNA.The properties and origin of MS brain pHERV-W ENV soluble antigen will allow a better understanding of the role of HERVs in MS pathogenesis.The present results anyhow pave the way to an accurate detection of the different forms of pHERV-W ENV antigen with appropriate conditions that remained unseen until now.

CRYSTAL STRUCTURE DETERMINATION OF 4-ZINC BOVINE INSULIN AT 1.9 (?) RESOLUTION

With the isomorphous difference Fourier method and the restrained least-squares re-finement technique, the crystal structure of 4- zinc bovine insulin in space group R_3 wasdetermined. The final crystallographic residual is 0. 19 at 1. 9A resolution. Based on therefined model, the structural mechanism for prolonged action of 4- zinc insulin is discussed.

Phase Relationship in Phenol-Insulin Crystal Growth System

Based on the crystal growth system of rhombohedral 2Zn-insulin,the phase transition ofinsulin crystals has been investigated with the phenol concentration as an independent component.The dia-gram of the phase relationship in this crystal growth system was established,and two points of phase transi-tion were found.The transition point Ⅰ indicates the phase transition between rhombohedral 2Zn-insulin crys-tal and rhombohedral 4Zn-insulin crystal,and these two phases coexist within a narrow region of phenol con-centration (0.028%-0.029% (g/ml)).Point Ⅱ at 0.76%-0.77% (g/ml) of phenol concentration showsthe phase transition between rhombohcdral crystal and monoclinic crystals,and a new phase of monocliniccrystal (B-form monoclinic insulin crystal) has been observed.This paper reports the diagram of phase rela-tionship obtained from our experiments,and analyses and discusses the dependence of phase transition of in-sulin crystals on phenol concentration in crystal growth system.

Crystal structural studies of destripeptide (B28-B30) insulin

Single crystals of destripeptide (B28-B30) insulin (DTRI) in three forms were obtained by hanging-drop vapor diffusion method. Form 1 belongs to P21 space group with cell parameters a-4.77 nm, b=6.19 nm, c=6.12 nm, β=110.3°. Form 2 belongs to P4122 or P4322 space group with cell parameters a= 6.45 nm, c=12.07 nm. Form 3 belongs to P212121 space group with cell parameters a=4.98 nm, b=5.16 nm, c=10.06 nm. The structure of form 1 crystal was determined by molecular replacement method and refined at 0.23 nm resolution. The R-factor of the final model is 18.8% with r.m.s. deviations of 0.001 5 nm and 3.3?for the bond lengths and the bond angles, respectively. Studies on the crystal structure show that the removal of B28 Pro has brought DTRI structural changes which made it dissociate more easily than native insulin although DTRI can still form a hexamer.

Self-assembly of L-tryptophan on Cu(111) studied by low-temperature scanning tunneling microscopy

The self-assembly of L-tryptophan on Cu(111) is investigated by an ultrahigh vacuum scanning tunneling microscope(STM) at 4.4 K.When deposited onto the substrate at around 120 K with a coverage of 0.1 monolayer,molecular trimers,tetramers,hexamers,and chains coexist on Cu(111).Then almost all molecules self-assemble into chiral hexamers after being annealed at room temperature.When increasing molecular coverage to the full layer,a new type of chain is observed on the surface.Based on the high-resolution STM images at sub-molecular level,we suggest that the L-tryptophan molecules are present in neutral,zwitterionic or anionic states in these structures.